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. 2013 Nov 26;110(48):19555-60.
doi: 10.1073/pnas.1318268110. Epub 2013 Nov 11.

Transmission of Multiple System Atrophy Prions to Transgenic Mice

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Free PMC article

Transmission of Multiple System Atrophy Prions to Transgenic Mice

Joel C Watts et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Prions are proteins that adopt alternative conformations, which become self-propagating. Increasing evidence argues that prions feature in the synucleinopathies that include Parkinson's disease, Lewy body dementia, and multiple system atrophy (MSA). Although TgM83(+/+) mice homozygous for a mutant A53T α-synuclein transgene begin developing CNS dysfunction spontaneously at ∼10 mo of age, uninoculated TgM83(+/-) mice (hemizygous for the transgene) remain healthy. To determine whether MSA brains contain α-synuclein prions, we inoculated the TgM83(+/-) mice with brain homogenates from two pathologically confirmed MSA cases. Inoculated TgM83(+/-) mice developed progressive signs of neurologic disease with an incubation period of ∼100 d, whereas the same mice inoculated with brain homogenates from spontaneously ill TgM83(+/+) mice developed neurologic dysfunction in ∼210 d. Brains of MSA-inoculated mice exhibited prominent astrocytic gliosis and microglial activation as well as widespread deposits of phosphorylated α-synuclein that were proteinase K sensitive, detergent insoluble, and formic acid extractable. Our results provide compelling evidence that α-synuclein aggregates formed in the brains of MSA patients are transmissible and, as such, are prions. The MSA prion represents a unique human pathogen that is lethal upon transmission to Tg mice and as such, is reminiscent of the prion causing kuru, which was transmitted to chimpanzees nearly 5 decades ago.

Keywords: bioluminescence imaging; neurodegeneration; proteinopathies; seeding.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Absence of spontaneous neurologic disease in hemizygous Tg(M83+/−:Gfap-luc) mice. Tg(M83+/−:Gfap-luc) mice are denoted as “+/−” and Tg(M83+/+:Gfap-luc) mice as “+/+.” (A) Kaplan–Meier curves for the appearance of signs of spontaneous neurologic dysfunction in Tg(M83+/−:Gfap-luc) (dashed line, n = 7) and Tg(M83+/+:Gfap-luc) (solid line, n = 8) mice. Only the Tg(M83+/+:Gfap-luc) mice developed spontaneous disease. (B) Sequential extraction of α-synuclein from the brains of two asymptomatic Tg(M83+/−:Gfap-luc) mice at 629 d of age (lanes 1–2) and two spontaneously ill Tg(M83+/+:Gfap-luc) mice at 549 d (lanes 3) and 389 d (lanes 4) of age. Brain homogenates were sequentially extracted with high salt (HS), high salt containing Triton X-100 (HS-T), SDS, and formic acid (FA) buffers and then analyzed by immunoblotting for human α-synuclein (Syn211 antibody). Aggregated, FA-extractable α-synuclein was only observed in the spontaneously ill Tg(M83+/+:Gfap-luc) mice. (CF) Immunohistochemical detection of phosphorylated α-synuclein in the brain of an asymptomatic Tg(M83+/−:Gfap-luc) mouse at 629 d of age (C and E) and a spontaneously ill Tg(M83+/+:Gfap-luc) mouse at 549 d of age (D and F). The reticular formation is shown in C and D and the brainstem in E and F. Phosphorylated α-synuclein deposits were only observed in the spontaneously ill Tg(M83+/+:Gfap-luc) mice. [Scale bar (in C): 50 μm (applies to D–F).] (G) Quantification of brain BLI signals (n = 6–14 per group) in asymptomatic Tg(M83+/+:Gfap-luc) and Tg(M83+/−:Gfap-luc) mice as well as Tg(Gfap-luc) mice (“non-Tg”) at the ages indicated, compared with spontaneously ill Tg(M83+/+:Gfap-luc) mice between 308 and 554 d of age (last BLI scans taken at 282–548 d, before the appearance of clinical symptoms). Brain BLI levels were significantly higher in spontaneously ill Tg(M83+/+:Gfap-luc) mice compared with either young Tg(M83+/+:Gfap-luc) mice (**P < 0.01) or aged Tg(M83+/−:Gfap-luc) mice (***P < 0.001), whereas levels were not significantly different (n.s.) between aged Tg(M83+/−:Gfap-luc) and Tg(Gfap-luc) mice.
Fig. 2.
Fig. 2.
Survival and BLI in Tg(M83+/−:Gfap-luc) mice inoculated with brain homogenate containing α-synuclein aggregates. (A) Sequential extraction of phosphorylated α-synuclein in brain homogenates used as inocula. Samples from human control, MSA (cases i and ii), and spontaneously ill TgM83+/+ (samples i and ii) brains were sequentially extracted with high-salt, high-salt containing Triton X-100, SDS, and formic acid (FA) buffers, and then the SDS and FA fractions were analyzed by immunoblotting. Aggregated, SDS- and FA-extractable phosphorylated α-synuclein was only observed in the MSA and TgM83+/+ samples. S129-phosphorylated α-synuclein was detected using the antibody EP1536Y. (B) Kaplan–Meier curves for the appearance of clinical signs of neurologic dysfunction in Tg(M83+/−:Gfap-luc) mice injected with inocula prepared from a human control brain (green line, n = 8), spontaneously ill TgM83+/+ mice (two independent samples, blue lines, n = 8 and 7), and MSA patients (two independent cases, red lines, n = 8 each). (C) BLI curves showed a significant increase (*P < 0.05) in TgM83+/+-inoculated animals (blue curve, n = 10) compared with uninoculated controls (black curve, n = 16) beginning at ∼150 d postinoculation. (D) MSA-inoculated Tg(M83+/-:Gfap-luc) mice at 84–112 d postinoculation exhibited higher brain BLI signals than control-inoculated mice at 356 d postinoculation.
Fig. 3.
Fig. 3.
Biochemical analysis of α-synuclein in the brains of inoculated Tg(M83+/−:Gfap-luc) mice. (A) Levels of detergent-insoluble phosphorylated α-synuclein were higher in the brains of clinically ill TgM83+/+-inoculated Tg(M83+/−:Gfap-luc) mice (191–250 d postinoculation) compared with age-matched, uninoculated mice. Two different inocula [M83 (i) and M83 (ii)] were prepared from two different spontaneously ill TgM83+/+ mice. Total levels of human α-synuclein are shown as a control. (B) Quantification of detergent-insoluble phosphorylated α-synuclein levels in the brains of clinically ill, TgM83+/+-inoculated Tg(M83+/−:Gfap-luc) mice and age-matched, uninoculated controls (n = 4 each). Phosphorylated α-synuclein levels were significantly higher in the inoculated mice (**P < 0.01, ***P < 0.001). (C) Levels of detergent-insoluble phosphorylated α-synuclein were higher in the brains of clinically ill Tg(M83+/−:Gfap-luc) mice that were inoculated with two different cases of MSA (88–110 d postinoculation) compared with control-inoculated mice (362 d postinoculation). Total levels of human α-synuclein are shown as a control. (D) Quantification of detergent-insoluble phosphorylated α-synuclein levels in the brains of clinically ill, MSA-inoculated Tg(M83+/−:Gfap-luc) mice and asymptomatic, control-inoculated mice (n = 4 each). Phosphorylated α-synuclein levels were significantly higher in the MSA-inoculated mice (**P < 0.01). (E) Sequential extraction of phosphorylated α-synuclein from the brains of Tg(M83+/−:Gfap-luc) mice inoculated with TgM83+/+ samples (clinically ill at 238 and 191 d postinoculation), control brain (asymptomatic at 362 d postinoculation), and MSA samples (clinically ill at 105 and 92 d postinoculation). For comparison, samples from asymptomatic, uninoculated Tg(M83+/−:Gfap-luc) mice at 259 d of age (None) are shown. Aggregated, FA-extractable phosphorylated α-synuclein was only observed in the TgM83+/+- and MSA-inoculated mice. For all immunoblots, S129-phosphorylated α-synuclein was detected using the antibody EP1536Y.
Fig. 4.
Fig. 4.
Widespread deposition of phosphorylated α-synuclein in the brains of inoculated Tg(M83+/−:Gfap-luc) mice. Immunohistochemical detection of phosphorylated α-synuclein in the brains of asymptomatic, control-inoculated Tg(M83+/−:Gfap-luc) mice (362 d postinoculation, left column) as well as clinically ill TgM83+/+-inoculated mice (194–209 d postinoculation, center column) or MSA-inoculated mice (88–105 d postinoculation, right column). The cortex is shown in AC, the thalamus in DF, the subthalamic nuclei in GI, the periaqueductal gray in JL, the reticular formation in MO, and the brainstem in PR. Phosphorylated α-synuclein deposits in the cell bodies of neurons and neuronal processes were only observed in the TgM83+/+- and MSA-inoculated mice. [Scale bar (in A): 50 μm (applies to all panels).]

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